1. Field of the Invention
This invention aims at enhancing drug bio-availability by providing an effective UGT2B inhibitor as well as a UGT2B enhancer for increasing the detoxification ability of individuals.
2. Description of the Prior Art
The drug metabolism process in human body, especially the metabolism of high fat-soluble drugs, includes two biotransformation steps: phase I reaction that catalyzes fat-soluble molecules to polarized molecules, and phase II reaction that produces highly polarized molecule through conjugation, such that the drugs can be metabolized efficiently and excreted to urine or feces.
The most common and important conjugation is glucuronidation by uridine diphosphate (UDP)-glucuronosyltransferases (refers to as UGTs; EC 2.4.1.17 hereafter).
The UGTs is one of the major enzymes in phase II reaction in human. It is now evident that UGTs have more than 110 isoenzymes. UGTs can catalyze the conjugation of UDP-glucuronic acid (UDPGA) and the endogenous fat-soluble compounds' chemical bonds, such as hydroxyl, sulfonyl, carboxylic acid, amine, or amide, to facilitate the O-glucuronidation, N-glucuronidation, or S-glucuronidation (King et al., 2000, Curr. Drug Metab., 1(2): 143-61), and thus enhances the polarity of the fat-soluble molecules.
According to a review article published by Radominska-Pandya et al (Drug Metab Rev. 31(4):817-99, 1999), most human UGTs belong to the UGT1A and UGT2B families. The UGT1A family consists of UGT1A1, UGT1A2P, UGT1A3-10, UGT1A11P and UGT1A12p, while UGT2B family consists of UGT2B4, UGT2B7, UGT2B10, UGT2B15 and UGT2B17.
In addition, UGTs posses extensive substrate specificity. The UGT1A and UGT2B metabolize different compounds. The UGT1A family mainly metabolizes phenolic compounds such as estrone, 2-hydroxyestrone, 4-nitrophenol, 1-naphthol, etc. with the involvement of bilirubin. The UGT2B family metabolizes steroid compounds such as androsterone, linoleic acid, etc. with the involvement of bile acids.
It was reported that UGTs can either enhance the bio-activity of some compounds or, under certain circumstances, transform some compound into toxic substances, such as morphine, steroids, bile acids and mid retinoids. (see Vore et al. (1983a) Life Sciences 32:2989-2993; Vore et al. (1983b) Drug Metabolism Reviews 14:1005-1019; Abbott and Palmour (1988) Life Sciences 43:1685-169). It was also reported that UGTs have involved in the activation of polycyclic aromatic hydrocarbons (PAH) and heterocyclic aromatic amines. (Munzel et al. (1996) Archives of Biochemistry and Biophysics, 355: 205-210; Bock et al. (1998) Advances in Enzyme Regulation, 38: 207-222).
UGTs can be found in several tissues including liver, kidney, bile duct, esophagus, stomach, intestine, rectum, ileum, jejunum, spleen, mammary gland, skin, lung, and brain. However, the distribution of various UGTs in human body differs by type. For instance, UGT2B7 exists mainly in esophagus, liver, intestine, colon, kidney, and spleen; while UGT1A1 can be found in liver, bile duct, stomach and colon (Tukey et al. (2000) Annu. Rev. Pharmacol. Toxicol., 40: 581-616. Review).
Studies by Burchell and Coughtrie (Burchell B and Coughtrie MW (1997) Environmental Health Perspectives 105: 739-747) found differences among individuals in their abilities to metabolize medicines, due to the genetic polymorphisms in UGT genes. Therefore, the information regarding the regulatory function of UGTs in individual's drug metabolism process is essential in evaluating a drug's potential pharmaceutical efficacy and its interaction with other drugs.
The UGTs is also an important detoxification system in human body. In addition to the endogenous fat-soluble compounds, the exogenous fat-soluble compounds can also become water-soluble through glucuronidation, and thus enhances the excretion of the exogenous fat-soluble compounds and maintains human body's normal detoxification function.
Therefore, the glucuronidation will be hampered by defected UGTs activities in individuals who suffered from liver diseases. Consequently, the liver's lower clearance rate in metabolizing drug will increase the toxic reaction and the rate of carcinogenesis in an individual with liver diseases.
According to literatures, butylated hydroxyanisole (BHA) (Buetler et al. (1995) Toxicology & Applied Pharmacology 135(1): 45-57) and pregnenolone-16α-carbonitrile (PCN) (Viollon-Abadie et al., 1999, Toxicology & Applied Pharmacology., 155(1):1-12) may activate UGT2B.
Before circulating through the entire body, most drugs that are absorbable to gastroenterological tract will enter the liver through portal circulation. This is the so-called “first-pass effect”. It had been confirmed that the ubiquitous UGTs in the intestine and the liver is one of the major enzymes that are necessary to the “first-pass effect” of the drug absorbance process. Such “first-pass effect” will stabilize a drug's bio-availability.
Owing to this phenomenon, the pharmacological scientists are aggressively looking for safe, effective, and reversible UGT inhibitors to apply to drugs with low bio-availability due to their fast metabolism, for the purpose of increasing their efficacy. Such a need is especially evident in oral medicines.
Studies in UGT inhibitors and their interactions with drugs have been conducted in recent year. Reported UGT inhibitors include silymarin (Venkataramanan et al. 2000, Drug Metabolism and Disposition 28: 1270-1273), quinoline (Dong et al., 1999, Drug Metabolism & Disposition 27:1423-1428), oltipraz (Vargas et al., 1998, Drug Metabolism & Disposition 26:91-97), tacrolimus (Zucker et al., 1999, Therapeutic Drug Monitoring 21:35-43), octyl gallate, apigenin, imipramine, clozapine, acetaminophen, and emodin (Radominska-Pandya et al., 1999, as mentioned earlier).
It was also reported that diazepam and flunitrazepam (FM2) can strongly inhibit the activity of UGT2B (Grancharov et al., 2001, harmacol Ther., 89(2):171-86).
Since the aforementioned UGT inhibitors are active drug ingredients by themselves and will induce prominent physical responses, they are not good candidates as drug absorption enhancers.
It was well recognized among those who are familiar with the techniques that a good UGT inhibitor for enhancing the bio-availability of drugs should at least posses the following three characteristics: (1) No or minimum pharmacological effect, except inhibiting UGT; (2) The inhibition should be reversible. In other words, UGTs should be able to restore its normal functions, after the inhibitors were excreted or metabolized; and (3) The efficacy of the inhibitor should be able to prominently lower the activity of UGTs in the intestine and the liver with a minimum dose.
It was known in recent years that grapefruit juice and certain components of other natural products, such as narigin, naringenin, hesperidine and other flavonoids, can inhibit some pharmacological activities.
US 6,121,23 depicts that by using essential oil, one can enhance the bio-availability of an oral medicine in the intestine of a mammal. The method involves co-administration of a therapeutic dose of the pharmaceutical compound and an essential oil or a component of essential oil where 10% inhibition was demonstrated with the presence of no more than 0.01 wt. of essential oil or a component of essential oil. In this US patent, it was also demonstrated that essential oil enhances the bio-availability of the drug through its inhibition of cytochrome P450.
Another study indicated that flavonoids compounds prepared from the liver of Long-Evants rat, such as naringenin, hesperetin, kaempferol, quercetin, rutin, flavone, α-naphthoflavone, and β-naphthoflavone can inhibit the metabolism of estrone and estradiol in microsomes (Zhu et al. 1998, J Steroid Biochem Mol Biol, 64(3-4): 207-15).
According to Chinese herbal medicine literature, due to its relatively milder toxicity than synthetic compounds, Chinese herbal enhancers (CHEs) were widely used in about 30%-75% of Chinese herbal medicine prescriptions. According to Japan's Food and Drug Administration, among 210 official Chinese herbal compound prescriptions, Glycyrrhizae radix is the most frequently used CHE (in 150 or 71.4% prescriptions), followed by Zingiberis (in 42.9% prescriptions) and Zizyphi fructus (in 31.9% prescriptions). In the Japanese National Formulary (2nd Edition), the most frequently used CHEs are Glycyrrhizae radix (71.4%), Zingiberis (42.9%), Holen (poria) (35.2%), Paeoniae radix (32.9%), Zizyphi fructus (31.9%) and Cinnamami cortex (29.5%).
Other than these frequently used CHEs, studies regarding other CHEs are rare. For the purpose of developing new UGT inhibitors, further investigations on some other CHEs are worthwhile. For instance, flavonoids-containing CHEs like Scutellariae radix (contains baicalin, wogonin, baicalein, skullcap-flavon I and wogoin glucuronide), Artemisiae cpillaris herba (contains capillarisin, cirsilineol, cirsimaritin, genkwanin, rhamrcocitrin); and terpenoids-containing CHEs like Alismatis rhizome (contains alisol monoacetate and triterpenoids), Moutan radicis cortex (contains paeoniflorin, oxypaeoniflorin and its benzoyl derivatives), Aconiti tuber (contains aconitine, mesaconitine, jesaconitine and atisine), Tragacantha (contains tragoside I and tragoside II), Persical semen (contains 24-methylenecycloartanol), Cimicifugae rhizome (contains cimigenol, cimigenol xyloside and its 12-hydroxyl derivatives and dahurinol).
Possible mechanisms of CHEs as enhancers of drug absorption include: (1) catalysts: new active ingredients may be derived from the cooking or the preparation process of Chinese herbal medicine; (2) carrier: carrying the drug's active ingredients going through barriers to reach its targets; (3) enzyme inhibitor: take UGT inhibitor as an example, if a drug's active ingredients cannot be absorbed by oral administration due to UGT metabolism, it may become orally absorbable by combining with a CHEs that lowers or restricts the “first-pass effect”.
This application's inventor found that glycyrrhizin in Glycyrrhizae radix and oleanolic acid and β-myrcene in Zizyphi fructus can enhance the partition coefficient in drugs like acyclovir, buprenorphine, or buprenorphine and hence increase these drug's transdermal permeation more than 1,000 percent in either in vitro or clinical trials. Reference: Dr. Oliver Yoa-Pu Hu's acyclovir Patent (Taiwan Patent No. 084682, U.S. Pat. No. 6,162,459, Japan Patent No. 2681881); buprenorphine Patent (Taiwan Patent No. 137835, U.S. Pat. No. 6,004,969); and piroxicam Patent (Taiwan Patent No. 133855).
New opioids drugs such as buprenorphine, nalbuphine and butorphanol were developed recently. They are classified as narcotic agonist-antagonist analgesics, due to their dual agonistic and antagonistic effects on opioids-receptors (Schmidt, W. K. et al, Drug Alcohol Depend. 14, 339, 1985). In addition to having high affinity to opioids-receptors, these dual-effect drugs can also be used as an antagonist to compensate for the drawbacks of narcotic analgesics, such as to lower their addictive effect and to drastically minimize their respiratory inhibition.
According to Schmidt et al (1985), nalbuphine possesses both the affinity to Kappa receptor (OP2) and the antagonist effect to Mu receptor (OP3). There is no obvious addiction or synergistic effect with only a slight respiratory inhibition, after a six-month continuous usage of nalbuphine. Therefore, in clinical trials, nalbuphine is safer than the traditional narcotic analgesics, and has exhibited an excellent therapeutic effect.
The drawback of this drug is its poor absorbability when delivered orally. In Goodman and Gillman's study, the bio-availability of nalbuphine is 11±4%, while the bio-availability of nalbuphine in animal is 2.7±0.4%, a shorter half-life. In current clinical pharmacology, the drug can only be administered through IV, not orally. The nalbuphine's pharmacokinetic studies indicate its half life through the excretion of liver is 5 hours, and about 7% of the drug are excreted to the urine in its original form (Birgit et al. 1996, Drug metabolism and Disposition, 25(1):1-4; Birgit et al. 1998, Drug Metabolism and Disposition, 26(1):73-77; and Richard et al. 1990, Clin. Pharmacol Ther., 47:12-19).
Published literature has proved that nalbuphine is mainly metabolized through UGT2B7 (Radominska-Pandya et al., 1999).
U.S. Pat. No. 6,004,969 depicts a transdermal delivery of buprenorphine. The method includes the delivery of a drug to patients that contains the following pharmaceutical ingredients: 1) about 0.8% of buprenorphine or its HCl salt; 2) about 10-20% of one or a combination of the following drug enhancers: 2-pinene, trans-cinnamic acid, β-myrcene or β-myrcene; and 3) about 79.2-89.2% of one or a combination of the following inert ingredients: stearyl alcohol, sodium carboxymethyl-cellulose, glycerol, cetyl alcohol, 1,3-propylene glycol, and water.
Studies regarding the use of Chinese herbal medicine in diseases treatment and prevention have become prominent in recent years. However, the issues about what are the herbal medicines that can inhibit UGT and how to apply them to the UGT-related therapeutic usages are still need to be addressed.
Besides, there are a lot of studies indicate that many pharmaceutical excipients might have effect on the activity of cytochrome P450. It is thought generally that most of the excipients are used to increase the volume, the solubility or stability and so on of drugs. However, such point of view might need to be changed.
Mountfield et al. (Mountfield et al., 2000) indicates at 2000 that some of the excipients influence CYP3A4 enzyme activity in microsomal enzyme experiment in vitro. Some other excipients are also reported in other researches, for example, PEG400 (Johnson et al., 2003), Cremophor RH40 (Wabdel et al., 2003) both have effect on the enzyme activity of CYP3A4.
Bravo et al. (Bravo et al., 2003) have studied the effect of surfactant on activity of enzyme. The medicine studied is colchicine that is metabolized by P-gp and CYP3A4 in vivo. The control group is colchicine dissolved in 0.9% NaCl solution, and the experimental group is colchicine dissolved in 5% solutol HS15-NaCl solution. The result shows that the concentration of experimental group is above two times of controlled group. The clearing rate of experimental group is obviously two folds lower than controlled group. And in-vitro hepatocyte experiment shows that 0.003% solutol HS15 inhibits clearing rate of colchicine. It is worth noted that this kind of surfactants are thought to destroy cell membrane in other studies, affect metabolism of cell (Silva et al., 2004), change the usage of co-factor in catalysis reaction of enzymes or interaction between substrate and enzyme. However, there is no evidence showing that solutol HS15 at such concentration has any impact on the intact of the cell under light microscope. Therefore, the detailed mechanism needs to be investigated in the future.
Generally speaking, the research and development of new medicine could be divided into several stages: New Chemical Entity (NCE), pre-clinical toxicity testing, pharmacological testing and finally, the clinical testing. Traditional chemical selection is to modify the structure on those who has pharmacological activity or extract the biological chemicals from plants. Unfortunately, most of these chemicals are fat-soluble compound, thus, different excipients are added to improve solubility and stability of these chemicals. For pharmaceutical industry, it is inevitable to use excipient. Hence, this invention not only studies the effect of using CHEs as UGT inhibitor and enhancer, but also the effect of excipients on enzyme activity of UGT in order to provide more UGT inhibitors and enhancers that are safe and effective.
In sum, the development of a safe, effective and reversible UGT inhibitor will enable the oral administration of high “first-pass effect” drugs, and will minimize the side effect and the dosage of highly variable drugs. In addition, the toxicity of the carcinogenic compounds caused by UGT activities can also be reduced.
Developing a safe, efficient and reversible UGT enhancer is also a desirable goal of the pharmaceutical industry. It will help patients with low clearance rate in drug metabolism due to reduced liver functions to metabolize the drugs, and enhance the detoxification function of the liver.